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20X SSC Buffer, pH 7.0
(20X Saline-Sodium Citrate Buffer)
Contains 3 M sodium chloride
0.3 M sodium citrate
DNase, RNase and protease-none detected
Catalog Number ML 012-01
Storage Temperature 15~30°C
SSC (Saline-Sodium Citrate) buffer is used for the attachment of nucleic acids on membranes for detection of certain nucleic acids or for measuring certain reactions of nucleic acids. SSC buffer is used in the following techniques; (1) Southern hybridization, (2) Northern hybridization, (3) Transfer and fixation of denatured RNA to uncharged nylone membranes at neutral pH, (4) Dot and slot hybridization of purified RNA, (5) Blocking agents for southern and northern hybridization. SSC buffer presents a high concentration of salts requiring for the attachment of nucleic acids on the membranes, such as nylon or nitrocellulose.
ML 012-01 contains 175.3 g of sodium chloride and 88.2 g of sodium citrate per 1 L of ultra pure water (ML 019-02). Before use, if necessary, dilute this 20X solution in ultra pure water to the desired concentrations, such as 10X, 6X, 2X, or 0.1X.
The liquid should be stored at 15~30°C. Deterioration of the liquid may be recognized by (1) precipitate or particulate matter throughout the solution, (2) cloudy appearance, (3) color change, and/or (4) pH change. The nature of supplements added may affect storage conditions and shelf life of the medium. Product label bears expiration date.
For In Vitro Use Only
Hybridization is a technique in which a single strand of nucleic acid associates with a complementary single strand of nucleic acid, forming a double strand. DNA is used as a template in Southern hybridization, while RNA is used as a template in Northern hybridization.
In 1975, a certain DNA fragment was detected on a nitrocellulose membrane using a specific DNA probe after transfer from a gel. Steps in this technique are as follows: (1) Digestion of DNA (genomic or plasmid) by restriction enzymes and fractionation by gel electrophoresis. (2) Denaturation of double strands to single strands in alkali condition. (3) Transfer of DNA fragments onto a nitrocellulose or a nylon membrane. (4) Fixation of DNA fragments by baking or UV-radiation. (5) Hybridization of radiolabeled or fluoroscence-labeled probe with DNA fragments. (6) Detection of probes hybridized with certain DNA fragments using appropriate methods.
Except the differences of DNA and RNA’s properties, this method is similar to the Southern hybridization. Steps in this technique are as follows: (1) Fractionation of RNA fragments by gel electrophoresis in the denaturated condition (treated with formaldehyde or glyoxal/DMSO). (2) Transfer of RNA fragments onto a nitrocellulose or a nylone membrane. (3) Fixation of DNA fragments by baking or UV-radiation. (4) Hybridization of radiolabeled or fluoroscence-labeled probe with DNA fragments. (5) Detection of probes hybridized with certain DNA fragments using appropriate methods.
Blocking agents for nucleic acid hybridization
Blocking agents prevent ligands from sticking to surfaces. They are used in molecular cloning to stop sonspecific binding of probes in Southern, northern, and western blotting. Without blocking agents, it would be impossible to detect anything but the strongest target macromolecules. As blocking agents, Denhardt’s reagent and BLOTTO (bovine lacto transfer technique optimizer) are used widely.
50X Denhardt’s Reagent
1% (w/v) Ficoll 400
1% (w/v) polyvinylpyrrolidone
1% (w/v) bovine serum albumin (fraction V)
5% (w/v) nonfat dried milk
0.02% sodium azide
Blocking agent (1)
6X SSC (or SSPE)
5X Denhardt’s Reagent
100 mg/ml denatured, sheared salmon sperm DNA
Blocking agent (2)
6X SSC (or SSPE)